A simple and cost‐effective method for studying anoxia tolerance in plants

Abstract Premise We developed a novel, cost‐effective protocol that facilitates testing anoxia tolerance in plants without access to specialized equipment. Methods and Results Arabidopsis thaliana and barley (Hordeum vulgare) seedlings were treated in airtight 2‐L Kilner jars. An anoxic atmosphere was generated using Oxoid AnaeroGen 2.5‐L sachets placed on in‐house, custom‐built wire stands. The performed experiments confirmed a higher sensitivity to low oxygen stress previously observed in anac017 A. thaliana mutants and the positive effect of exogenous sucrose on anoxia tolerance reported by previous studies in A. thaliana. Barley seedlings displayed typical responses to anoxia treatment, including shoot growth cessation and the induction of marker genes for anaerobic metabolism and ethylene biosynthesis in root tissue. Conclusions The results validate the novel method as an inexpensive, simple alternative for testing anoxia tolerance in plants, where access to an anaerobic workstation is not possible. The novel protocol requires minimum investment and is easily adaptable.

Oxygen deficiency (hypoxia) and oxygen absence (anoxia) are abiotic stresses with dramatic effects on plant physiology. They result in an "energy crisis" in plant cells and tissues due to restricted respiration (Gibbs and Greenway, 2003;Huang et al., 2008). The ability to cope with hypoxia and/or anoxia is particularly relevant in the context of plant flooding tolerance due to the relatively low solubility of oxygen in water and the slow diffusion of gases in water compared to air (Jackson, 1985;Voesen et al., 2006). Depending on the height of the water column produced, the extent of flooding may range from soil waterlogging to complete submergence, when the aerial plant tissues are covered by water (Jackson and Colmer, 2005). Furthermore, the adverse effects of these stresses can be exacerbated by ice encasement, where the water flooding the field freezes and the rate of exchange of respiratory gases drops to extremely low levels, requiring anoxia tolerance for plant survival (Andrews, 1996). Indeed, a recent meta-analysis estimated that flooding-induced global crop yield losses may exceed 30% (Tian et al., 2021), and this is projected to increase in many regions due to increasingly volatile weather associated with climate change (Hov et al., 2013;Lehtonen et al., 2014). Consequently, there is an urgent need to further elucidate the molecular mechanisms that underpin plant responses to oxygen deprivation (Zahra et al., 2021) and develop floodresilient cultivars.
Most studies of anoxia tolerance in the model species Arabidopsis thaliana (L.) Heynh. have been performed using an enclosed anaerobic workstation to provide an oxygen-free environment for plant incubation, such as the Anaerobic System Model 1025 (Forma Scientific, Marietta, Ohio, USA) or 1 Person Hypoxic Glove Box (Coy Laboratory Products, Grass Lake, Michigan, USA) (Kursteiner et al., 2003;Banti et al., 2008;Tagliani et al., 2020). A similar setup was also used to study anoxia tolerance in rice seedlings (Lasanthi-Kudahettige et al., 2007). However, not all laboratories have access to a commercial anaerobic workstation, and purchasing one is a significant investment (in excess of €20,000, dependent on specifications) that may be unaffordable to many research groups. An alternative approach reported in the literature involves placing plants in airtight jars flushed with a nitrogen gas stream and/or premixed air (Konkina et al., 2021). However, this also requires access to anaerobic jars with gas valves or another self-contained anaerobic system, and thus may incur significant costs.
Herein, we describe and validate a novel airtight jar method for testing anoxia tolerance in plants that is reliable, cost-effective, and easily accessible. Airtight Kilner jars were purchased from a local household and kitchen supplies store (less than €10 per 2-L jar) and used in combination with Oxoid AnaeroGen 2.5-L sachets (Thermo Fisher Scientific, Basingstoke, United Kingdom), an affordable atmospheregenerating product frequently used by microbiologists for the cultivation of anaerobic bacteria (~€60 per pack of 10 sachets, depending on the geographic location/supplier). We successfully used the protocol to corroborate findings previously obtained for A. thaliana with other methods, such as use of anaerobic workstations and submergence tolerance testing (Banti et al., 2008;Bui et al., 2020;Meng et al., 2020). This suggests that the developed method can be successfully used to dissect the mechanisms underlying anoxia tolerance in this model species, where a multitude of genetic resources are readily available. Additionally, we demonstrate the suitability of the method for inducing anoxia responses in barley (Hordeum vulgare L.) seedlings and propose that it can be applied to identify genotypes and cultivars resilient to this abiotic stress.

Experimental setup
Anoxia treatment was performed in 2-L airtight round Kilner Clip Top jars with rubber seals (product code 0025.493; Rayware Group, Liverpool, United Kingdom) purchased from a local Homebase Ltd. store (Dublin, Ireland). Galvanized 1.5-mm steel wire and duct tape were used to construct simple stands (see Figure 1A for an example) for placing the Oxoid AnaeroGen 2.5-L (AN0025, Thermo Fisher Scientific) sachets at the top part of the jar. Before the treatment, a thin layer of petroleum jelly was applied to the rubber seals of the jar to ensure an airtight lock. The Oxoid Resazurin Anaerobic Indicator (BR0055B, Thermo Fisher Scientific) was placed on the wall of the jar following manufacturer's instructions. To initiate anoxia treatment, the AnaeroGen foil sachets were opened and the AnaeroGen paper sachets were folded in half and immediately placed on the wire stand inside the Kilner jars. (See Appendix 1, section 1.1, for a complete list of required materials.) The jars were closed 45-60 s after opening the AnaeroGen foil sachets. AnaeroGen paper sachets contain ascorbic acid and activated carbon, which react with air, rapidly absorbing oxygen and producing carbon dioxide (Thermo Fisher Scientific, 2022). The Anaerobic Indicator consists of a cotton strip impregnated with pink resazurin solution that is reversibly reduced to colorless dihydroresorufin when oxygen becomes limited. The gradual change of the Anaerobic Indicator cotton strip from pink to white confirmed generation of an anoxic atmosphere within 2 h ( Figure 1B, C). As the AnaeroGen sachets generate some heat due to an exothermic reaction between the active component ascorbic acid and oxygen, the sachets were also placed in control jars, but without F I G U R E 1 Outline of the developed protocol for anoxia treatment. (A) Place plant material (e.g., Arabidopsis thaliana or barley seedlings) on 35-mm Petri dishes, wire stand, anaerobic indicator, and AnaeroGen sachet in the 2-L Kilner jar (25.5 mm × 13.7 mm × 12.4 mm). (B) Close the jar 45-60 s after the AnaeroGen sachet has been opened. (C) As anoxia develops, the anaerobic indicator (*) will change from pink to white. an airtight seal, to ensure that the observed results of the anoxia treatment were not caused by an increase in temperature. See Appendix 2, section 2.1, for a detailed summary of the experimental protocol.

Arabidopsis thaliana
Arabidopsis thaliana wild-type (Col0) and anac017-1 homozygous T-DNA insertion knockout line were used for experiments. Anac017 was a kind gift from Jim Whelan (LaTrobe University, Australia), derived from a T-DNA insertion line originally obtained from the European Arabidopsis Stock Centre, generated by the Salk Institute Genomic Analysis Laboratory (Alonso et al., 2003) and previously characterized by Ng et al. (2013). Seeds were surface sterilized for 20 min in 20% (v/v) commercial bleach (final concentration of NaOCl approximately 1%), washed four times with sterile distilled water, and plated in 35-mm Petri dishes containing half-strength Murashige and Skoog basal salt medium (2.15 g L −1 , M0221.0050; Duchefa Biochemie, Haarlem, The Netherlands), 0.6% agar (A1296; Sigma Aldrich, Darmstadt, Germany), supplemented with sucrose (S5391, Sigma Aldrich) or glucose (G0802.1000, Duchefa Biochemie) at concentrations of 30 mM or 90 mM (pH 5.6-5.8) (see below). The dishes were sealed with Leukopor tape (02453-00; Essity, Hull, United Kingdom) that allows gaseous exchange but prevents moisture loss and placed at 4°C in the dark for 3 d before being moved to a 22°C constant temperature growth room for germination at the indicated light regime. See Appendix 1, section 1.2, for a list of required materials for growth of A. thaliana seedlings and Appendix 2, section 2.2, for protocol details.

Barley
Seeds of winter barley cultivars (Cavalier and Siberia) were a kind gift from Dr. Susanne Barth (Teagasc, Carlow, Ireland). The seeds were surface sterilized using a 20% commercial bleach (v/v) solution for 10 min, washed at least four times with sterile distilled water to remove any bleach residue, and placed in 90-mm Petri dishes lined with moistened 85-mm filter paper (approximately 20 seeds per Petri dish). The Petri dishes were sealed with Leukopor tape and placed at 4°C in the dark for 7 d to achieve synchronized seed germination. The seeds were allowed to germinate under a 16 h light/8 h dark regime at 22°C for 3 d before seedlings were used for experiments. See Appendix 1, section 1.2, for a list of required materials for growth of barely seedlings and Appendix 2, section 2.2, for protocol details.

Arabidopsis thaliana
Four dishes with A. thaliana seedings were placed inside each Kilner jar, with Leukopor tape removed, for a 3-d (72h) anoxia treatment. The anoxia treatment was interrupted after 72 h by opening the jars; the dishes were then resealed with Leukopor tape and the seedlings allowed to recover. The seedling age, light regime, and recovery time for the individual F I G U R E 2 Protective effect of sucrose, but not glucose, on anoxia survival in Arabidopsis thaliana. Col0 seedlings were treated according to a protocol adapted from Banti et al. (2008). Seeds were germinated in the dark on 35-mm Petri dishes supplemented with 30/90 mM of sucrose or glucose, subjected to a 3-d anoxia treatment in the dark, followed by a 7-d recovery period in 16 h light/8 h dark conditions. (A) Representative images of plants after the 7-d recovery period. (B) Graph representing mean seedling survival (±SEM) at 30/90 mM treatments. Three experimental repeats (n = 10) were performed; P value obtained using Student's t-test is indicated. Scale bar = 1 cm. experiments are indicated in Figures 2 and 3, and the protocol details are provided in Appendix 2, section 2.2.

Barley
The 3-d-old barley seedlings were transferred to 35-mm Petri dishes filled with soil substitute vermiculite saturated with distilled water. One seedling was placed per Petri dish, and four Petri dishes were placed inside each Kilner jar (two Petri dishes with each cultivar). The jars were closed and placed in the growth room for 24 h under a 16 h light/8 h dark regime at 22°C. On the following day, the jars were opened, seedling shoot length was recorded (Day 0, prior to anoxia treatment), and anoxia treatment was initiated. The anoxia treatment was interrupted after 24 h by opening the jars to allow re-oxygenation by ambient air. The shoot growth of individual seedlings was measured immediately after the end of anoxia treatment, as well as after the first and second day of recovery. The details of the experimental protocol are summarized in Appendix 2, section 2.2. The samples of root tissue (approximately 50 mg fresh weight) were collected from the seedlings immediately after anoxia treatment for quantifying gene expression of PDC (pyruvate decarboxylase) and ACO (1-amino cyclopropane 1-carboxylic acid oxidase). The total RNA was isolated using an RNeasy Mini Plant Kit (Qiagen, Hilden, Germany) with the on-column DNase I digestion step included. The RNA concentration and purity were measured using a NanoDrop Spectrophotometer (Thermo Fisher Scientific), and complementary DNA (cDNA) was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Quantitative PCR (qPCR) was performed using KAPA SYBR FAST qPCR Master Mix (KAPA Biosystems, Wilmington, Massachusetts, USA) in the ViiA 7 qPCR system (Thermo Fisher Scientific) using protocol and primer sequences described by Luan et al. (2018).
F I G U R E 3 The anac017 mutant shows impaired anoxia tolerance. Col0 and anac017 seeds were germinated in 16 h light/8 h dark at 22°C on 35-mm Petri dishes supplemented with 1% sucrose. Ten-day-old plants were subjected to a 3-d anoxia treatment, followed by a 4-d recovery period. Subsequently, plant survival and percentage of damaged leaves per seedling were recorded. (A) Representative images of Col0 and anac017 plants after a 4-d recovery period. (B) Graphs representing mean seedling survival and percentage of damaged leaves (±SEM). Six experimental repeats (n = 5) were performed. Untreated (control) seedlings displayed 100% survival and no leaf damage. P values indicate statistically significant difference between genotypes (Student's t-test). Scale bar = 1 cm.

Statistical analyses
The obtained data were analyzed (Student's t-test) using IBM SPSS Statistics for Windows software (version 27.0; IBM, Armonk, New York, USA).

Applicability of the airtight jar method for studying anoxia responses in the model species Arabidopsis thaliana
We used the developed method to successfully replicate the results from a previous study that utilized an enclosed anaerobic workstation for investigating anoxia tolerance in this crucial model species (Banti et al., 2008). Banti et al. (2008) reported a protective effect of exogenous sucrose, but not glucose, on anoxia tolerance in A. thaliana. We adapted their protocol to the airtight jar method to confirm that similar results can be obtained without the use of an anaerobic workstation. Indeed, anoxia treatment (72 h) of 4-d-old, dark-germinated Col0 seedlings resulted in no survival on solid medium supplemented with 30 mM sucrose, in contrast to 53% survival on medium supplemented with 90 mM sucrose ( Figure 2). However, a similar effect has not been observed when the growth medium was supplemented with glucose, with no seedlings surviving anoxia treatment at both 30 mM and 90 mM concentration, also in agreement with Banti et al. (2008). In addition, we used the developed method to estimate the anoxia tolerance of mitochondrial retrograde signaling mutant, anac017, that has been previously reported to show impaired submergence tolerance (Bui et al., 2020;Meng et al., 2020) (Figure 3). For these experiments, plants were grown for 10 d (16 h light/8 h dark) prior to anoxia treatment, as the submergence sensitivity phenotype of anac017 has been reported to be enhanced with seedling age (Bui et al., 2020). As anticipated, treatment of Col0 and anac017 plants revealed higher anoxia sensitivity of anac017 seedlings, which showed significantly lower treatment survival and an increased level of leaf damage (Figure 3). These results highlight that the airtight jar method offers a simple protocol to study the effect of growth conditions and genotype on anoxia tolerance in the model species A. thaliana and can be easily adapted to meet experimental requirements (e.g., in terms of age of treated plants, chemical supplementation, light and temperature regime).

Applicability of the airtight jar method for studying anoxia responses in barley
Barley is relatively sensitive to flooding and soil waterlogging (Setter and Waters, 2003;de San Celedonio et al., 2014). The identification of barley germplasm with increased tolerance to oxygen deprivation, and characterization of the genetic mechanisms underlying this tolerance, is therefore urgently required. Herein, we tested the developed protocol on two winter barley cultivars, Cavalier and Siberia ( Figure 4). As expected, the anoxia treatment resulted in almost complete shoot growth cessation in both cultivars ( Figure 4A). Shoot growth resumed during an anoxia recovery period, with Cavalier showing faster shoot growth recovery compared to Siberia. This is in line with a recently published study where prolonged (15 d) waterlogging treatment, followed by six weeks of recovery, significantly reduced the plant height of Siberia but had no effect on the Cavalier cultivar (Miricescu et al., 2021). Moreover, ACO, the gene involved in ethylene biosynthesis (Houben and Van de Poel, 2019), and PDC, which catalyzes the first step in the ethanolic fermentation pathway (Jackson and Colmer, 2005), were significantly induced by the anoxia treatment in the roots of both cultivars ( Figure 4D). Previously, upregulation of these genes in barley root tissue was suggested to be a critical factor driving the flooding responses in this species (Luan et al., 2018). Additionally, during optimization of the protocol for barley we also confirmed that there was no growth rate reduction when AnaeroGen sachets were placed in the jars without an airtight seal (compared to jars without sachets), which further suggested that the effects of the applied treatment were not due to the generated heat but resulted from oxygen deprivation.
Collectively, the results suggest that the developed protocol for anoxia treatment: (i) facilitates imposing rapid and severe oxygen deprivation stress on crop seedlings in a repetitive and reliable manner, (ii) allows quick detection of differences between studied cultivars that mirror those observed in other experimental systems, and (iii) induces a gene expression profile typical for response to low oxygen conditions. Consequently, the airtight jar method presented in this study has the potential to advance research on the mechanisms underlying responses to oxygen deprivation at an early plant growth stage, as well as the identification of tolerant germplasm, in barley and other crop species. It needs to be highlighted that while the method can be used for a variety of species, the size of the airtight Kilner jars makes the protocol suitable for studying anoxia responses in small species, or in plants at early developmental stages, such as barley seedlings, as described herein.

CONCLUSIONS
We developed a novel protocol for studying anoxia tolerance in plants using airtight Kilner jars and Oxoid AnaeroGen anaerobic gas-generating sachets. The low cost, simplicity, and adaptability of the proposed experimental setup suggest that it would be of interest to researchers studying oxygen deprivation responses in plants who have no access to anaerobic workstations. One consideration while using the developed protocol is that when an AnaeroGen sachet is placed in an airtight jar, the oxygen is rapidly absorbed, leading to simultaneous generation of carbon dioxide, with resulting CO 2 levels of 9-13% (ThermoFisher Scientific, 2022). Similarly, CO 2 is found to accumulate in waterlogged soils (Setter and Waters, 2003), and toxic concentrations of CO 2 in soil were shown to negatively affect plant growth in previous studies. For example, prolonged exposure to soil CO 2 concentrations of 41-65% reduced root water absorption, chlorophyll, starch content, and total biomass (He et al., 2019). It is therefore possible that the effects observed here are partially linked to the high CO 2 levels generated in the jars. This increase in CO 2 concentration needs to be taken into account when planning experiments involving the airtight jar method, especially when prolonged anoxia treatment is applied. In the future, additional optimizations of the method that could potentially reduce CO 2 accumulation in the jars are possible, for example, based on absorption of CO 2 by dry soda lime.
In this study, we demonstrated that the established airtight jar method is suitable for studying the effect of growth conditions and genotype on anoxia tolerance in the model species A. thaliana. Herein, we applied anoxia treatments of up to 72 h, but if required this period could be extended, with the used resazurin indicator making it possible to monitor continued maintenance of anaerobic conditions. Moreover, the method is transferable between species and can be used to study anoxia-induced physiological and molecular responses, as well as to identify differences in cultivar tolerance in crops such as barley. In conclusion, the described method has a strong potential to advance both basic and applied research and is particularly relevant considering the urgent need to increase our understanding of how plants cope with oxygen deprivation stress.

ACKNOWLEDGMENTS
The authors thank Dr. Susanne Barth (Teagasc, Carlow, Ireland), who gifted us the barley cultivars, and Jim Whelan F I G U R E 4 Response of barley seedlings to anoxia treatment. Cavalier and Siberia seeds were germinated in 16 h light/8 h dark at 22°C on 85-mm moistened filter paper inside 90-mm Petri dishes. Four-day-old plants were subjected to a 24-h anoxia treatment, followed by a 48-h recovery period. (A) Barley shoot growth rate (mm/day) during the anoxia treatment and recovery period. Cavalier and Siberia, two winter barley cultivars, were tested. Bars represent the mean of three replicates (±SEM) (n = 8). Crt = control, T = Treatment; P value determined using Student's t-test is indicated. (B) A 24-h anoxia treatment induces shoot growth cessation in barley seedlings. Scale bar = 1 cm. (C) Cavalier shows faster shoot growth recovery following the treatment; control and treated Siberia and Cavalier seedlings after a 24-h anoxia treatment plus a 24-h recovery period are shown. Scale bar = 1 cm. (D) Expression of ACO and PDC genes induced by the 24-h anoxia treatment in both cultivars. Bars represent the mean of three experimental replicates (±SEM) (n = 2). Indicated P values were determined using Student's t-test on log 2 -transformed relative gene expression data. NS = not significant.
(LaTrobe University, Australia), who kindly shared the anac017 mutant seeds to enable this work. We thank the Environmental Protection Agency and Irish Research Council for PhD funding provided to O.L.S. (grant number: irc80800bad9bbf74695ba9c1f3c2982cea). Open access funding provided by IReL.